1. Field of the Invention
This invention relates to the manufacture and use of recombinant albumin fusion proteins and combinations thereof, and particularly to yeast expressed fusion proteins formed between human albumin and bioactive molecules such as therapeutic proteins and peptides, and more particularly to yeast expressed fusion proteins formed between human albumin and cell proliferation stimulatory factor (CPSF), such as, blood cell-stimulatory factors, erythropoietin (EPO), interleukins (ILs), stem cell factor (SCF), thrombopoietin (TPO), granulocyte colony stimulating factor (G-CSF), and granulocyte macrophage colony stimulating factor (GM-CSF).
2. Description of Related Art
1. Albumin
Albumin is a soluble, monomeric protein which comprises about one-half of the blood serum protein. Albumin functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones and plays a role in stabilizing extracellular fluid volume. Mutations in this gene on chromosome 4 result in various anomalous proteins. Albumin is a globular un-glycosylated serum protein of molecular weight 65,000. The human albumin gene is 16,961 nucleotides long from the putative ‘cap’ site to the first poly(A) addition site. It is split into 15 exons which are symmetrically placed within the 3 domains that are thought to have arisen by triplication of a single primordial domain. Albumin is synthesized in the liver as pre-pro-albumin which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce the secreted albumin. HSA has 35 cysteins; in blood this protein monomer has 17 disulfide linkage (Brown, J. R. “Albumin structure, Function, and Uses” Pergamon, New York, 1977). HSA is misfolded when produced intracellularly in yeast without its amino terminal secretion peptide sequence. This conclusion is based on its insolubility, loss of great than 90% of its antigenicity (as compared to human-derived HSA), and formation of large protein aggregates. At present albumin for clinical use is produced by extraction from human blood. The production of recombinant albumin in microorganisms has been disclosed in EP 330 451 and EP 361 991.
Albumin is a stable plasma transporter function provided by any albumin variant and in particular by human albumin. HSA is highly polymorphic and more than 30 different genetic alleles have been reported (Weikamp L, R, et al., Ann. Hum. Genet., 37 219-226, 1973). The albumin molecule, whose three-dimensional structure has been characterized by X-ray diffraction (Carter D. C. et al., Science 244, 1195-1198, 1989), was chosen to provide the stable transporter function because it is the most abundant plasma protein (40 g per liter in human), it has a high plasma half-life (14-20 days in human, Waldmann T. A., in “Albumin Structure, Function and Uses”, Rosenoer V. M. et al (eds), Pergamon Press, Oxford, 255-275,1977), and above all it has the advantage of being devoid of enzymatic function, thus permitting its therapeutic utilization at high dose.
2. Interleukin-11 (IL-11)
Human IL-11 (Paul et al. (1990), Pro. Natl. Acad. Sci. 87:7512) has been identified in medium conditioned by primate bone marrow-derived stromal cells. IL-11 is expressed in cells of mesenchymal origin, such as stromal fibroblasts, fetal lung fibroblasts and trophoblasts.
IL-11, also called adipogenesis inhibitory factor (AGIF), acts on hematopoietic progenitor cells and stromal cells (Kawashima, I., et al., Progress in Growth Factor Research, 4,191 1992). The mature molecule is a non-glycosylation protein, 178aa in length, and has an apparent molecular weight 23 KD (as determined by SDS-PAGE). Human IL-11 gene consists of five exons and four introns and was mapped on chromosome 19 at band 19q13.3-q13.4. IL-11 exhibits a primary structure unrelated to that of known cytokines, but it often acts similar to other cytokines, notably IL-6. IL-11 is a pleiotropic growth factor effecting hematopoietic and non-hematopoietic cells, often in synergy with interleukins, colony stimulating factors, or stem cell factor. In hematopoietic cells, IL-11 can enhance megakaryopoiesis, stimulate early and intermediate myeloid progenitor cells, initiate proliferation of dormant hematopoietic progenitor cells, and stimulate T-cell-dependent development of antibody-secreting B-cells. In non-hematopoietic cells, IL-11 can inhibit adipogenesis, and mediates the hepatic acute phase response. IL-11 stimulated the production of erythrocytes was reported only by Quesniaux, V F J., et al., Blood, 80, 1218 (1992).
Human IL-11 (e.g., NEURMEGA®, manufactured by America Home Products Company) has been approved for clinical trials in the United States for directly stimulating the proliferation of hematopoietic stem cells and megakaryocyte progenitor cells and inducing megakaryocyte maturation, resulting in increased platelet production. It has been used for the prevention of severe thrombocytopenia and the reduction of the need for platelet transfusion following myelosuppressive chemotherapy.
3. Erythropoietin (EPO)
Erythropoietin (EPO) is a glycoprotein that is the principle regulator of red blood cells growth and differentiation (U.S. Pat. No. 5,547,933). Erythropoiesis, the production of red blood cells, occurs continuously throughout the human life span to offset cell destruction. Erythropoiesis is a very precisely controlled physiological mechanism enabling sufficient numbers of red blood cells to be available in the blood for proper tissue oxygenation, but not so many that the cells would impede circulation. The formation of red blood cells occurs in the bone marrow and is under the control of the hormone EPO.
EPO is an acidic glycoprotein (˜30,400 Daltons) produced primarily by the kidney and is the principal factor regulating red blood cell production in mammals. Renal production of EPO is regulated by changes in oxygen availability. Under conditions of hypoxia, the level of EPO in the circulation increases and this leads to increased production of red blood cells. The over-expression of EPO may be associated with certain pathophysiological conditions. Polycythemia exists when there is an overproduction of red blood cells (RBCs). Primary polycythemias, such as Polycythemia vera, are caused by EPO-independent growth of erythrocytic progenitors from abnormal stem cells and low to normal levels of EPO are found in the serum of affected patients.
On the other hand, various types of secondary polycythemias are associated with the production of higher than normal levels of EPO. The overproduction of EPO may be an adaptive response associated with conditions that produce tissue hypoxia, such as living at high altitude, chronic obstructive pulmonary disease, cyanotic heart disease, sleep apnea, high-affinity hemoglobinopathy, smoking, or localized renal hypoxia. In other instances, excessive EPO levels are the result of production by neoplastic cells. Cases of increased EPO production and erythrocytosis have been recorded for patients with renal carcinomas, benign renal tumors, Wilms' tumors, hepatomas liver carcinomas, cerebellar hemangioblastomas, adrenal gland tumors, smooth muscle tumors, and leiomyomas.
Deficient EPO production is found in conjunction with certain forms of anemias. These include anemia of renal failure and end-stage renal disease, anemias of chronic disorders [chronic infections, autoimmune diseases, rheumatoid arthritis, AIDS, malignancies], anemia of prematurity, anemia of hypothyroidism, and anemia of malnutrition. Many of these conditions are associated with the generation of IL-1 and TNF-, factors that have been shown to be inhibitors of EPO activity. Other forms of anemias, on the other hand, are due to EPO-independent causes and affected individuals show elevated levels of EPO. These forms include aplastic anemias, iron deficiency anemias, thalassemias, megaloblastic anemias, pure red cell aplasias, and myelodysplastic syndromes.
The amount of erythropoietin in the circulation is increased under conditions of hypoxia when oxygen transport by blood cells in the circulation is reduced. Hypoxia may be caused by loss of large amounts of blood through hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged unconsciousness, or various forms of anemia. In response to tissues undergoing hypoxic stress, erythropoietin will increase red blood cell production by stimulating the conversion of primitive precursor cells in the bone marrow into proerythroblasts which subsequently mature, synthesize hemoglobin and are released into the circulation as red blood cells. When the number of red blood cells in circulation is greater than needed for normal tissue oxygen requirements, erythropoietin in circulation is decreased.
EPO may occur in three forms: alpha-, beta and asialo-EPO. The alpha- and beta-forms differ slightly in carbohydrate components, but have the same potency, biological activity and molecular weight. The asialo-form is an alpha- or beta-form with the terminal carbohydrate (sialic acid) removed. EPO is present in very low concentrations in plasma when the body is in a healthy state wherein tissues receive sufficient oxygenation from the existing number of erythrocytes. This normal low concentration is enough to stimulate replacement of red blood cells which are lost normally through aging. See generally for references, Testa, et al., Exp. Hematol., 8(Supp. 8), 144-152 (1980); Tong, et al., J. Biol. Chem., 256(24), 12666-12672 (1981); Goldwasser, J. Cell. Physiol., 110(Supp 1), 133-135 (1982); Finch, Blood, 60(6), 1241-1246 (1982); Sytowski, et al., Exp. Hematol., 8(Supp 8), 52-64 (1980): Naughton, Ann. Clin. Lab. Sci., 13(5), 432-438 (1983); Weiss, et al., Am. J. Vet. Res., 44(10), 1832-1835 (1983); Lappin, et al., Exp. Hematol., 11(7), 661-666 (1983); Baciu, et al., Ann. N.Y. Acad. Sci., 414, 66-72 (1983); Murphy, et al., Acta. Haematologica Japonica, 46(7), 1380-1396 (1983); Dessypris, et al., Brit. J. Haematol, 56, 295-306 (1984); and, Emmanouel, et al., Am. J. Physiol., 247 (1 Pt 2), F168-76 (1984).
Because EPO is essential in the process of red blood cell formation, the hormone has potential useful application in both the diagnosis and the treatment of blood disorders characterized by low or defective red blood cell production. See, generally, Pennathur-Das, et al., Blood, 63(5), 1168-71 (1984) and Haddy, Am. Jour. Ped. Hematol./Oncol., 4, 191-196, (1982) relating to erythropoietin in possible therapies for sickle cell disease, and Eschbach, et al. J. Clin. Invest., 74(2), pp. 434-441, (1984), describing a therapeutic regimen for uremic sheep based on in vivo response to erythropoietin-rich plasma infusions and proposing a dosage of 10 U EPO/kg per day for 15-40 days as corrective of anemia of the type associated with chronic renal failure. See also, Krane, Henry Ford Hosp. Med. J., 31(3), 177-181 (1983).
Prior attempts to obtain erythropoietin in good yield from plasma or urine have proven relatively unsuccessful. Complicated and sophisticated laboratory techniques are necessary and generally result in the collection of very small amounts of impure and unstable extracts containing erythropoietin. Genetically engineered EPO (U.S. Pat. No. 5,547,933) has been expressed in Chinese Hamster Ovary cell line (CHO). It has been approved for administration on clinical trials by FDA and now manufactures by Amgen Inc. under the name of EPOGEN.
It has been estimated that the availability of erythropoietin in quantity would allow for treatment each year of anemias of 1,600,000 persons in the United States alone. See, e.g., Morrison, “Bioprocessing in Space—an Overview”, pp. 557-571 in The World Biotech Report 1984, Volume 2:USA, (Online Publications, New York, N.Y. 1984). Recent studies have provided a basis for projection of efficacy of erythropoietin therapy in a variety of disease states, disorders and states of hematologic irregularity: Vedovato, et al., Acta. Haematol, 71, 211-213 (1984) (beta-thalassemia); Vichinsky, et al., J. Pediatr., 105(1), 15-21 (1984) (cystic fibrosis); Cotes, et al., Brit. J. Obstet. Gyneacol., 90(4), 304-311 (1983) (pregnancy, menstrual disorders); Haga, et al., Acta. Pediatr. Scand., 72, 827-831 (1983) (early anemia of prematurity); Claus-Walker, et al., Arch. Phys. Med. Rehabil., 65, 370-374 (1984) (spinal cord injury); Dunn, et al., Eur. J. Appl. Physiol., 52, 178-182 (1984) (space flight); Miller, et al., Brit. J. Haematol., 52, 545-590 (1982) (acute blood loss); Udupa, et al., J. Lab. Clin. Med., 103(4), 574-580 and 581-588 (1984); and Lipschitz, et al., Blood, 63(3), 502-509 (1983) (aging); and Dainiak, et al., Cancer, 51(6), 1101-1106 (1983) and Schwartz, et al., Otolaryngol., 109, 269-272 (1983) (various neoplastic disease states accompanied by abnormal erythropoiesis).
4. Granulocyte Colony Stimulating Factor (G-CSF)
Granulocyte colony stimulating factor (G-CSF) is produced by monocytes and fibroblasts. It stimulating granulocyte colony formation, activates neutrophils, differentiates certain myeloid leukemic cell lines and is a potent activator of mature granulocytes (Metcalf D., cell, 43, 5, 1985; Groopman, J. E., Cell,. 50, 5 1987);. Nature human G-CSF is a 19.6 KD glycoprotein having 177 amino acids (Souza, L. M et al., Science, 232, 62, 1986). Human and murine GCSF share approximately 75% amino acid sequence homology and have biology cross-reactivity (Morstyn, G., and Burgess, A., Cancer Res., 48, 5624, 1988). The biological activity of recombinant human G-CSF was measured in a cell proliferation assay using NFS-60 cells (Shurafuji, N et al., Exp. Hematol., 17, 116, 1989). Human G-CSF has been brought to the market under the name of NEUPOGEN® by Amgen, Inc.
5. Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)
Granulocyte-Macrophage colony stimulating factor (GM-CSF) induces myeloid progenitor cells from bone marrow to from colonies contains macrophages and granulocytes in semisolid media. GM-CSF also acts upon mature macrophages, eosinophils and nutrophils to stimulate various functional activities (Mazur, E., and Cohen, J., Clin Pharmacol. Ther., 46, 250, 1989; Morstyn, T G., and Burgess. A., Cancer Res., 48, 5624, 1988). GM-CSF is an acidic glycoprotein {18-22 KD human (Wong, G., et al., Science, 228, 810, 1986), 23 KD mouse (Metcalf, D., Blood, 67, 257, 1986)} which binds to high affinity receptors on GM-CSF sensitive cells. Although human and mouse GMCSF share 54% amino acid sequence homology, their biological actions are species-specific (Metcalf, D., Blood, 67, 257, 1986). Other growth factors and CSFs modulate receptor binding or actions of GM-CSF (Nicola, N., Immunol. Today, 8, 134, 1987). The proliferative activity of human GMCSF is tested in culture using human TF-1 cells (Kitamura, T., et al., J. cell Physiol., 140, 323, 1989). Human GM-CSF has been brought to the market under the name of LEUKINE® by Immunex, Inc
6. Macrophage Colony Stimulating Factor (M-CSF)
Macrophage Colony Stimulating Factor (M-CSF) is produced by monocytes, fibroblasts and endothelial cells. It stimulates the formation of macrophage colonies (Metcalf, D., Blood, 67, 257, 1986), enhances antibody-dependent cell mediated cytotoxicity by monocytes and macrophages (Mufson, R. A. et al., Cellular Immunol., 119, 182, 1989), and inhibits bone resorption by osteoclasts (Hattersley, G., et al., J. Cell Physiol., 137, 199, 1988). M-CSF is glycoprotein and appears in a few different molecular weight forms due to variation in glycosylation. The peptide has 159 amino acids (Kawasaki, E. S., et al., Science, 230, 291, 1985).
7. Thrombopoietin (TPO)
Thrombopoietin (TPO), the ligand for the receptor encoded by the c-Mpl proto-oncogene, acts as a stimulator of the development of megakaryocyte precursors of platelets. Similar to erythropoietin, TPO leads to an increase in number of circulating platelets. TPO affects the entire thrombopoietic process, with stronger effects in the later stages. Other thrombopoietic cytokines include Stem cell factor (SCF), IL-3, IL-6 and IL-11.
TPO is an approximately 35 KD polypeptide of 335 amino acid. However, due to glycosylation the protein has an apparent molecular weight of 75 KD in SDS-PAGE. The precursor form of TPO consists of 356 amino acids. To generate the mature TPO (335aa), the precursor cleaves a 21 amino acids signal peptide. Human, mouse and dog TPO shows 69-75% amino acid homology. The biological activities of recombinant human TPO was measured in a cell proliferation assay using MO7e cells.
8. Interleukin-3 (IL-3)
Interleukin -3 (IL-3) is one of a large and growing group of growth factors which support the proliferation and differentiation of hematopoietic progenitors as well as cells committed to various myeloid lineages in vitro and in vivo. Human IL-3 has 133 amino acids in mature protein and the glycosylation is not necessary for biological activity in vitro and in vivo. The homology between human and murine IL-3 is considerably less. The initial studies on the biology and biochemistry of IL-3 shows that among the well characterized hematopoietic growth factors, IL-3, is the only factor to be predominantly, if not exclusively, produced by activated T cell in normal cells in mice (Ihele and Weinstein, 1986) as well as in human (Yang and Clark 1998). The structure of IL-3, and the structure and location of its gene, are very much like those of a number of the hematopoietic growth factors and suggest that IL-3 is a member of an evolutionarily related family of growth factors. In preclinical and clinical trials, the most prominent and consistent effect of I1-3 in vivo is a significant increase in the absolute neutrophil count (ANC). In vitro IL-3, in combination with other cytokines such as stem-cell factor, IL-6, IL-1, IL-11, G-CSF. GM-CSF, erythropoietin (EPO), or Thrombopoietin (TPO) induces the proliferation of colony-forming units granulocyte-macrophage (CFU-GM), CFU-Eo, CFU-Baso, burst-forming units-erythroid (BFU-E), colony-forming units-megakaryocyte (CFU-MK) and colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) in semisolid medium, and it stimulates the proliferation of purified CD34+ cells in suspension culture (Eder, et al., Stem Cell, 15:327-333, 1997).